Enhanced Neuropeptide Profiling via Capillary Electrophoresis Off

Jul 22, 2008 - An off-line interface incorporating sheathless flow and counter-flow balance is developed to couple capillary electrophoresis (CE) to m...
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Anal. Chem. 2008, 80, 6168–6177

Enhanced Neuropeptide Profiling via Capillary Electrophoresis Off-Line Coupled with MALDI FTMS Junhua Wang, Mingming Ma, Ruibing Chen, and Lingjun Li* School of Pharmacy and Department of Chemistry, University of Wisconsin—Madison, 777 Highland Avenue, Madison, Wisconsin 53705-2222 An off-line interface incorporating sheathless flow and counter-flow balance is developed to couple capillary electrophoresis (CE) to matrix-assisted laser desorption ionization Fourier transform mass spectrometry (MALDI FTMS) for neuropeptide analysis of complex tissue samples. The new interface provides excellent performance due to the integration of three aspects: (1) A porous polymer joint constructed near the capillary outlet for the electrical circuit completion has simplified the CE interface by eliminating a coaxial sheath liquid and enables independent optimization of separation and deposition. (2) The electroosmotic flow at reversed polarity (negative) mode CE is balanced and reversed by a pressure-initiated capillary siphoning (PICS) phenomenon, which offers improved CE resolution and simultaneously generates a low flow (1 month), which is important when repetitive experiments need to be performed. Other features of the CA membrane joint include the zero dead volume, good electrical conductivity, and easily reproducible fabrication without the need for extensive training or special apparatus. A well-fabricated porous CA joint should possess the following characteristics: (1) Care should be taken to prevent the clogging of the porous joint by CA. This was tested by flushing the capillary with water or CE buffer at 0.5 psi. A fluent flow of approximately 200 nL/min seen from the column outlet verifies the good connection of the joint. (2) No leakage. This was tested by flushing the capillary with air while keeping the joint in water and no bubbles should appear, verifying that the CA coating was uniform and no leakage was observed. (3) Good electrical conductivity. This was inspected by performing electrophoresis between the joint and the capillary inlet with buffers. A nice linear relationship (46) http://www.innovagen.se/custom-peptide-synthesis/peptide-propertycalculator/peptide-property-calculator-notes.asp. (47) Winzor, D. J. J. Chromatogr., A 2003, 1015, 199–204.

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between current (0 ∼ 70 µA) and voltage (0 ∼ 30 kV) was obtained with 0.5% ammonium acetate buffer, pH 4.5 as running buffer (Figure S-1, Supporting Information). Capillaries that meet the above criteria are ready for use and can usually be stored for more than 6 months without losing performance. The CA joint is stable in the presence of 25% (v/v) organic additives in the running buffer such as methanol and ACN but could be damaged by less than 10% acetone solution. PICS and the Flow Rate under Different Polarity Modes. In contrast to the conventional integrated CE systems where capillary siphoning is considered or controlled48 to a negligible level, on this custom-built CE device the siphoning flow was observed as a steady large flow lasting for 2-4 h once initiated by the micropressure. Briefly, we applied a micropressure to start the siphoning inside the capillary, and the pressure was retracted prior to the CE run; thus, no external pressure was applied during the separation, indicating that the bulk flow was maintained by siphoning only. Therefore, “pressure-initiated capillary siphoning” is fundamentally different from the terms such as “pressuredriven” or “pressure-assisted” because this specific counter-flow CE technique via siphoning is initiated by a pressure but relies on siphoning in the capillary to maintain its flow. Unlike pressuredriven flow that would produce a parabolic flow leading to reduced resolution, the flow profile produced by PICS is not well understood. A complete understanding of the fluidic pattern of siphoning-dominated bulk flow in the presence of EOF would require further investigation by some special techniques such as whole column imaging49 detection50 or computer simulation.51 The PICS flow rate (υs) was determined to be ∼75 nL/min (n ) 3) by using ammonium formate buffer (50 mM, 10% ACN, pH 3.5, 25 °C) with the height difference (∆h ) hinlet - houtlet) of 7.5 cm. When electrophoresis was started under a voltage of ±18 kV, the flow rates of CE stream changed to ∼96 nL/min (n ) 3) and ∼61 nL/min (n ) 3), corresponding to the flow in positive mode CE (υs + υeof) and negative (reversed polarity) mode CE (υs υeof), respectively. Note that the evaporation of the liquid at the capillary outlet and inside the pipet tip was not taken into account for the flow rate measurement; therefore, the determined flow rate can be viewed as an “apparent” flow rate. Further studies will be performed to determine the extent of evaporation and its effect on the CE experiment. Another important issue of concern is the gas formation at the electrodes, as described previously in a comprehensive review on CE-MS.9 Several factors including the CE current, flow rate, and the buffer pH change would affect the amount of generated bubbles in CE. In our CE assembly (Figure 1), the electrode is positioned about 2 mL (∼3.5 cm in distance) away from the capillary inlet (see Figure S-2 for the detailed schematic drawing of the CE inlet). This is a fairly large distance as compared to conventional CE in which the electrode and the capillary inlet are usually put together in a small vial. Furthermore, the current was controlled to a low level (∼20 µA) by applying a low electric field and using a low concentration buffer, which minimized the production of hydrogen gas at the (48) Jussila, M.; Palonen, S.; Porras, S. P.; Riekkola, M.-L. Electrophoresis 2000, 21, 586–592. (49) Fang, N.; Li, J.; Yeung, E. S. Anal. Chem. 2007, 79, 5343–5350. (50) O’Grady, J. F.; Noonan, K. Y.; McDonnell, P.; Mancuso, A. J.; Frederick, K. A. Electrophoresis 2007, 28, 2385–2390. (51) Kim, J. B.; Britz-McKibbin, P.; Hirokawa, T.; Terabe, S. Anal. Chem. 2003, 75, 3986–3993.

inlet end. Therefore, the bubble formation was not taken into account in the PICS technique reported here. The siphoning flow was several times greater than the EOF and resulted in the migration of all of the analytes toward the capillary outlet. The significant suppression of the flow rate of EOF was mainly due to two factors. First, the EOF mobility (µeof) was very low when using a low pH (pH 3.5) buffer solution, which was about 5.0 × 10-5 cm2 V-1 s-1, 8-9 times lower than that at high pH (e.g., pH 9.0).52 Second, the electric field strength (E ) 18 kV/75 cm) was also very low in comparison with a routine CE experiment. The theoretical EOF velocity (υeof ) µeofE) under this condition was ∼14.4 nL/min, which matched well with the experimental result. On the other hand, the unique construction of the CE setup produced siphoning free from backpressure because the capillary outlet was completely open in contrast to the format being surrounded with buffer in conventional CE. The flow balance was successfully achieved inside the capillary with the PICS technique, by adjusting the voltage and the ∆h to control the flow rate toward two ends. It was shown that the highest positively charged peptide was kept in the capillary for much longer time (over 35 min) than the negatively charged ones, resulting in greatly improved resolution of the CE separation. The siphoning-assisted flow counterbalance and sheathless deposition offers several attractive features such as simplicity for construction and steady but reduced flow rate (and thus increased mass sensitivity) for fraction collection and deposition. Evaluation of the Method with a Complex Mixture of Neuropeptides. A collection of 25 neuropeptides (NPs) with concentrations of 10-6-10-7 M was selected for the evaluation of this method (Table 1, for the q/M1/2 and Mf. values, see the discussion below). About 50 nL of samples were injected into the capillary for electrophoresis under both positive (+16 kV) and negative (-15 kV) modes of CE in the presence of the PICS flow. Running buffer was a cocktail of ammonium formate (100 mM)/ H2O/ACN with a ratio of 5:4:1, at pH 3.9. The fractions were collected every 30 s for a total of 40 min followed by MALDI FTMS analyses of these off-line deposited fractions. As expected, the separation under positive mode CE with PICS was much faster than that under negative mode, whereas the latter separation provided much better resolution for the mixture components. The improvement of resolution also has an effect on sensitivity enhancement. With the same sampling volume, separation buffer, and MS detection settings, the MS signal intensities of the peptides under reverse polarity mode CE-PICS were found to be about 5-fold higher than that under positive mode. As a result, all 25 NPs were detected in reverse polarity mode CE but 4 of the 25 NPs were not detected under positive CE mode because of inadequate ion abundance. One possible reason was due to longer and more effective focusing of the peptides in the capillary and better separation in the reverse polarity CE mode which reduced possible analyte suppression from coeluting peaks. Sensitivity improvement was evident with S/N > 200 for peptides at 1 × 10-7 M. The limit of detection of 1.5 nM was obtained for 75 amol of peptide from a 50 nL volume of solution at S/N ) 3. Linear Fitting of q/M1/2 Values vs Migration Time (t). The classical semiempirical formula in electrophoresis reveals a linear (52) Frazier, R. A.; Ames, J. M.; Nursten, H. E. Capillary Electrophoresis for Food Analysis: Method Development. Frazier, R. A., Ames, J. M., Nursten H. E., Eds; Royal Society of Chemistry Press: Cambridge, U.K., 2000; p 5.

correlation between the electrophoretic mobility (me) and the q/M1/2 value (where q is a peptide’s net charge, M is the molecular mass).38,53 Based on this correlation, it is possible to evaluate electrophoretic migration behavior37 and to study structural modifications and conformational changes of peptides and proteins with CE.39 The studied NPs were plotted as q against migration time (t) in Figure 2, left panel. The closed circles (b) represent the peptides being separated using a positive mode CE with PICS. The open circles (O) represent those separated with negative mode CE. The migration order was observed linearly related to the net charges, as extracted and shown in the inset below. It was observed that the larger me the peptide has, the more quickly it elutes in CE when operating in positive mode. Therefore, the migration time (t) of a peptide is inversely proportional to its me in the positive CE. In contrast, the t value is proportional to its me and accordingly proportional to its q/M1/2 value in the negative CE mode. These observations are plotted in Figure 2(a-d) showing the relationships of the q and q/M1/2 values and CE migration time under negative CE. It is noted that the pyroglutamylated peptide corazonin, pQTFQYSRGWTNa (MH+ ) 1369.7) migrates very early in the negative CE and elutes very late in the positive CE together with other acidic peptides such as the orcokinin family peptides. The calculated net charge at pH 3.9 is +2 if the N-terminal pGlu-modification is neglected, whereas the observed migration behavior indicates a -0.2 net charge for the pyroglutamylated peptide, slightly higher than the orcokinin family peptides (-0.33). Other known pyroglutamylated peptides were also observed to elute earlier during the negative CE separation, as is shown later in the results section. Figure 2b depicts the linear fitting of q/M1/2 values for all the 25 peptides against migration time (q/M1/2-t), a fairly linear correlation is obtained (R2 ) 0.903). With the removal of the badly deviated spot (MH+ ) 1369.7), the correlation improves to R2 ) 0.979 (Figure 2c), which is consistent with the classical semiempirical relationship. It is observed that the fitting with only q against t (Figure 2a) also shows a linear correlation with even better R2 (0.98) than the q/M1/2-t curve, indicating a direct linearity between q and t, which is independent of the molecular mass, M. The peptides with the earliest (n ) 2) and latest (n ) 2) migration time were used for another q/M1/2-t linear fitting, as shown in Figure 2d, yielding a line that is very close to that with the remaining 24 peptides included in Figure 2c. This result suggests that the acquired fitting curve with a greatly reduced number of peptides deviates only slightly from that with all peptides included. Such a simplified method should be useful for plotting the fitting curves in real sample analysis where the q/M1/2 values for most of the peptides are unknown. Since only limited data is needed to generate the fitting curve, it is often possible to identify a few known peptides from both ends of the electrophoretic migration order. Therefore, it is applicable for complex extract analysis, provided that the regressive validation of the fitting curve with all of the assigned peptides can give a satisfactory matching. This approach offers an increased confidence for peptide identification and was employed for the following real sample analysis. Peptide Migration Behavior Matching Evaluation. A matching factor (Mf.) is defined here as Mf. ) q/M1/2cal/(ktobs + b) (53) Offord, R. E. Nature 1966, 211, 591–593.

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Figure 2. Distribution of the experimental data of the peptides and linear fittings of (a) q and (b-d) q/M1/2 against t under negative CE (where q is net charge of peptide, M is molecular mass, t is migration time). Parts a and c are fittings with the removal of modified peptide (MH+ ) 1369.7) as compared to part b. (d) Fitting with two peptides in the earliest and latest eluted fractions, respectively.

according to the fitting curve for the peptides’ migration behavior matching evaluation (where q/M1/2cal is the calculated q over M1/2 of the peptide, tobs is the observed migration time, k and b are the slope and intercept for the fitting line, respectively). The Mf. values of the model peptides from Figure 2c,d are given in the Mf.(c) and Mf.(d) columns in Table 1, respectively. As seen, in both cases, most Mf. values of the peptides are around 1.0, the theoretical value, indicating that the fitting line matches perfectly with the peptides’ experimental distribution. Statistical analysis was performed with SAS software version 9.1 according to these data, average values of 1.032 ± 0.138 and 1.039 ± 0.135 were obtained for the Mf.(c) and Mf.(d) columns, respectively. Note that the corazonin (MH+ ) 1369.7) was identified as an outlier from statistical analysis due to its exceptionally large Mf. values (Mf.(c) ) 7.3, Mf.(d) ) 9.27). Another peptide, allatostatin IV, DRLYSFGLamide (MH+ ) 969.5, Mf.(c) )1.4, Mf.(d) ) 1.5) was also observed to deviate substantially from the confidence interval (CI, p < 0.05) of 0.76 ∼ 1.30. These elevated matching values (or matching errors) indicate the peptides’ earlier elution in CE as compared to their predicted times (tobs < tpre). This deviation often stems from post-translational modifications (PTMs) occurring on a given peptide (e.g., pQTFQYSRGWTNa) or the presence of specific amino acid residue (see discussion below), presenting a useful tool for elucidation of unique features of peptides. Enhanced Profiling of Neuropeptides from Tissue Extracts of C. borealis. We have previously employed numerous MS-based strategies including direct tissue MALDI MS and capillary LC coupled to tandem MS for neuropeptide discovery and characterization.42,43,54–56 To date, more than 250 neuropeptides from various decapod crustacean model organisms have been identified, with some being commonly present in several tissues, while others uniquely present in a specific tissue. However, numerous putative neuropeptides remain unidentified and their relevant physiological actions are unknown due to the low concentrations, extreme chemical diversity, and PTMs of these trace-level signaling molecules.57 (54) Fu, Q.; Goy, M. F.; Li, L. Biochem. Biophys. Res. Commun. 2005, 337, 765– 778. (55) Fu, Q.; Tang, L. S.; Marder, E.; Li, L. J. Neurochem. 2007, 101, 1099– 1107. (56) Ma, M.; Chen, R.; Sousa, G. L.; Bors, E. K.; Kwiatkowski, M. A.; Goiney, C. C.; Goy, M. F.; Christie, A. E.; Li, L. Gen. Comp. Endrocrinol. 2008, 395–409.

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Compared to capillary LC, CE consumes much less sample. For a typical LC-MS/MS experiment, often 50-100 animals’ tissues are pooled and homogenized for peptide extraction that are used for subsequent LC fractionation. In contrast, the minimal sample consumption of CE enables the use of tissue from a single animal or a few animals. In addition, the sample preparation procedure is simplified when coupling CE to MALDI MS detection, due to its tolerance to salts and denaturants. The reconstituted tissue extracts containing about 5 µL of supernatant were used for CE injection. In each run, ∼50 nL of the sample from pooled C. borealis POs (n ) 1 or 4), brains (n ) 5), or CoGs (n ) 6) were injected, respectively, for analysis by off-line CE and MALDI FTMS. CE fractions of PO extract were collected with an interval of 30 s for 50 min. A total of 62 fractions were found to contain peptide signals. The obtained CE-MALDI MS data set is displayed as a twodimensional (2D) plot in Figure 3A. A total of 160 putative peptide peaks were detected in the MALDI spectra, 43 peaks could be assigned to known NPs by searching the home-built database and eight peaks were assigned to the known NPs fragments or adduct. Reconstructed CE-MALDI electropherograms of these identified NPs are shown in Figure S-3. Nine SORI-CID fragmentation spectra were obtained on the fractions with high ion abundance, with one representative fragmentation spectrum shown for the peptide with a precursor mass of 1030.5 in Figure 3B. For comparison, the crude PO extract was desalted and concentrated by ZipTipC18, and a 0.5 µL aliquot of the processed extract was loaded and analyzed directly by MALDI FTMS. The direct analysis allowed identification of 19 peptides. Comparisons of mass spectra obtained using these two methods are shown in Figure 4. Three spectra of individual CE fractions are shown in Figure 4B-D. Although 10-fold less sample was used for CE-MALDI MS than for the direct MALDI MS mixture analysis, the former experiments with CE separation allowed detection of significantly more peptides with up to 10-fold improved sensitivity. This improvement in peptide coverage was mainly due to alleviation of signal suppression from the complex mixture by incorporating the CE separation step. In some cases, overlapping peptides close in mass can be better separated in the CE process. Furthermore, as is shown in Figure S-3, the intensity varies greatly (over 300-fold) from peptide to peptide, indicating a wide dynamic (57) DeKeyser, S. S.; Li, L. Anal. Bioanal. Chem. 2007, 387, 29–35.

Figure 3. (A) Bubble mapped 2D plot of the off-line CE-MALDI MS data. The bubble size is related to ion abundance of a peptide. Larger bubbles indicate more intense peptide signals. SORI-CID spectra are recorded from spots a to h indicated by arrows, with the precursor masses listed in the margin. (B) Fragmentation ion spectrum of pEGFYSQRYamide (MH+ ) 1030.5) with sequence-specific fragment ions labeled.

Figure 4. Comparison of the MS spectra of direct MALDI analysis and three of the CE fractions (a total of 43 peptides were identified by the CE-MS method). (A) 19 peptides (a-s) are identified with direct MALDI MS analysis. (B) Spectrum of fraction 18, with two identical (c and i) and three additional (u, v, w) peptides identified as compared to (A). (C) Spectrum of fraction 53, with six identical (a, b, l-n, p) and four additional (x, y, z, aa) peptides identified. (D) MS spectrum of fraction 57, with six identical (e, g, h, l, m, p) and two additional (aa, ab) peptides identified. The absolute intensity is shown on the right of each spectrum. The asterisks show unidentified peptides.

range of the complex sample. However, this quantitative information was masked in the direct MALDI MS analysis. Duplicate analyses of a single PO extract sample were carried out to evaluate the reproducibility of the method. Repetitions of the same extract conducted in a time span of 2 weeks showed very similar results in both spectral patterns and peptide intensities (see Figure S-4). Similar analytical results were obtained with the brain and CoG extracts (data not shown). These results were

combined together into a table (see Table S-1) for subsequent analysis and comparison. For more confident identity assignment, the mass error (Er.) and peptide electrophoretic behavior matching factor (Mf.) were introduced for peptide assignment from more than 220 detected peaks. Formaldehyde Labeling of PO Extracts. In addition to comparing the peptide migration matching factor, chemical derivatization represents an alternative method to give compleAnalytical Chemistry, Vol. 80, No. 16, August 15, 2008

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Table 2. Representative Examples of the Peptide Assignment Via Off-Line CE-MALDI MSa PO P

obsd MH+

N

(p)

1 2 3 4 5 6 7 8 9

965.548 1260.661 966.528 1486.724 1030.465 1369.635 969.486 1608.719 940.503

+ + +

+

10

1104.608

11 12 13 14 15 16

1602.851 1474.659 1502.701 1532.718 1547.674 1563.703

(s)

Br

CoG

Er.

Mf.

theoret

sequence

family

+ +

5.4 0.4 1.2 -4 -9 -13

1.01 1.11 2.4/D 2.4 6/pE 8/pQ

NRNFLRFa SGKWSNLRGAWa DRNFLRFa GFKNVEMMTARGFa pEGFYSQRYa pQTFQYSRGWTNa

+ +

+ + + +

-8.7 3.3 -1.3 1.7

1.57 1.05 0.83 1.05

+ + + + +

+ + + + +

-1 6.6 11 -1

0.99 1.01 1.02 1.03

965.5428 1260.6596 966.5268 1486.7294 1030.4741 1369.6522 Unk Unk 940.51116 940.49993 1104.6095 1104.6061 Unk 1474.65973 1502.69103 1532.70161 1547.67611 Unk

RFamide AST-B RFamide

+

+ + + + + + +

+

+

+

+ + + + + + +

+ + + + + + +

+ +

RFVGGSRYa QRAYSFGLa AQPSMRLRFa GAHKNYLRFa NFDEIDRSGFGFA NFDEIDRSGFGFV NFDEIDRSSFGFV NFDEIDRSSFGFN

RYamide Corazonin

AST-A RFamide Orco-LP Orco-LP Orco-LP Orco-LP

a Mass underlined, the unidentified peptides. Samples are from tissue(s) of pericardial organs (PO), brain (Br), and commissural ganglia (CoG), respectively. The columns under PO: the N column, normal sample without isotopic formaldehyde labeling; the (p) column, peptides detected as pairs with +28/+32 Da mass shift upon labeling; the (s) column, peptides detected as unlabeled single peaks. “+” shows that the peptides are detected in the samples. Er., the mass error in ppm. Mf., the custom defined matching factor of the peptide for observed and predicted migration time in CE. AST-A, -B: allatostatins A and B types. Orco-LP: orcokinin-like peptide. See the text for the details of these data analysis.

mentary information for neuropeptide identification. The Nterminal isotopic dimethylation reaction58 with formaldehyde was used to label the peptides prior to CE separation. The labeling with formaldehyde-H2 (FH2, light) and formaldehyde-d2 (FD2, heavy) can produce peaks with +28 and +32 Da mass shifts, respectively. In combining with CE, this protocol can provide increased confidence for peptide identification. For example, an observed single peak (without the expected mass shift) after the labeling reaction indicates that the peptide is N-terminally blocked, which is consistent with the observation of a unique earlier migration behavior in CE. On the other hand, a pair of peaks with 4.02 Da (+28/+32 mass shifts after derivatization) difference suggests that the original peptide is N-terminal free or N-terminally blocked but contains one lysine (K). Peptide Identity Assignment. As seen in Table 2, 16 representative peptides were selected from approximately 230 peaks to illustrate the procedure of peptide assignment used in the CE off-line MALDI MS experiment. Typically, four cases were considered for the identity assignment. Case 1: Highly confident assignment of the peptides (no. 1-3 in Table 2) with high mass measurement accuracy (Er. e 5.0 ppm) and good CE migration matching factor (Mf. ≈ 1.0) (for the Mf. value shown as 2.4/D, see the discussion below for the modified Mf. values for these peptides). The assignment of peptide 4 is incorrect due to the greatly deviated Mf. value (2.4). Case 2: Assignment of the pyroglutamylated peptides 5 and 6 according to the evidence of “earlier migration” in CE and nonreactivity to formaldehyde labeling as a result of N-terminal blockage. This information together with the SORI-CID MS/MS spectrum (Figure 3B), verified the pyroglutamylation modification on the N-terminus (E) of peptide 5, pEGFYSQRYa (MH+ ) 1030.5). Peptides 7 and 8 are two suspected examples of similar structures with amino acid sequences yet to be determined. Case 3: Assignment of the correct identity from two sequences with very close masses, either by Er. and Mf. values (peptide 9) or via a labeling reaction (peptide (58) Fu, Q.; Li, L. Anal. Chem. 2005, 77, 7783–7795.

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Figure 5. Coelution of four orcokinin-like family peptides in CE, with a similar abundance distribution in both C. borealis brain and CoG extracts. A novel low abundance peptide MH+ 1563.7 is observed to coelute with other members of the orcokinin peptide family; the isotopic peaks are shown in the inset.

10). For example, the assigned peptide QRAYSFGLa (MH+ ) 940.5) has a better match with the observed mass and Mf. value. In the case of peptide assignment for GAHKNYLRFa, both +28/ +32 and +56/+64 Da mass shifted pairs are observed in the spectra, indicating a peptide sequence with free N-terminus and a lysine (K) residue. Peptide 11 is an unknown peptide with the labeled pairs also detected by CE-MS. Case 4: Peptides belonging to a specific family that share significant sequence homology (peptides 12-15) are observed to comigrate or elute closely in CE. As shown in Figure 5, four orcokinin-like peptides coeluted in the CE separation and were found to be with a similar abundance

distribution in both brain and CoG samples. Another putative novel member of the orcokinin peptide family with even lower abundance also eluted together with the four peptides, with the identity yet to be de novo sequenced in a future study. Similarly, by comparing eluting profiles of reconstructed electropherograms of the 43 identified peptides (Figure S-3), it was observed that members from the same peptide family often comigrate and peptide families were eluted in the following order with the reversed polarity CE mode: pGlustructure > orcokinin-like peptide >AST-A > AST-B > RYamide > RFamide. Some peptides with more specific acidic (D, E) or basic (K, R) amino acid residues will exhibit more pronounced deviations in the migration order, as discussed above. Overall, the incorporation of CE fractionation has added another dimension of important information to increase the confidence of peptide assignment and also exhibited great potential for novel peptide discovery. Earlier Migration Peptides and PTM Characterization. In Table 2, highly increased Mf. values, as denoted with 6/pE and 8/pQ, were observed for several pyroglutamylated peptides. According to the definition of the Mf., the increased value indicates a lower observed net charge (q) as compared with the calculated one using the equation and also suggests an earlier migration in CE than the predicted time. As we know that in the pGlu-structure, the free amine group (sNH2) and the carboxyl (sCOOH) side chain of the E or Q forms a cyclic amide (sCONHs) at the N-terminus, which has blocked the protonation of both groups and thus leads to the lower net charge in an acidic environment. Other identified peptides in Table 2, P3, DRNFLRFa (MH+ ) 966.5) and in Table S-1, DVRTPALRLRFa (MH+ ) 1342.8) with the Mf. values of 2.4/D and 1.82/D, also exhibited great deviations and indicated slightly earlier migration than the predicted time with calculation. As observed above for allatostatin IV, DRLYSFGLa, the increased Mf. value was congruously found to occur in peptides with similar structures that are C-terminally amidated and contain an acidic residue (D) at the N-terminus. A modified net charge calculation method is therefore proposed for those and other similar peptides being subsequently identified in all samples (including others in Table S-1). The resulting q value is in close match to the experimental value by subtracting a basic residue such as K (or H, R) from the sequence (if there has one) when carrying out the calculation with the equation. These findings are

believed to be useful for the construction of predictive models of peptide migration behavior based on the same calculation method.37,59 CONCLUSIONS In summary, we have described an improved design of CE off-line coupled to MALDI FTMS and its successful application for neuropeptide analysis. Improved neuropeptidome coverage was achieved with less sample consumption and enhanced ability for prediction and characterization of novel neuropeptides and their corresponding peptide families. Over 220 putative peptide peaks were detected with a wide dynamic range from the crude extract of several pooled C. borealis neuronal tissues. Among these, 70 neuropeptides that belong to 10 families were identified with increased confidence by incorporating CE separation. The CE fractionation shows great potential for the global analysis of neuropeptides from complex samples by providing effective preconcentration, desalting, and separation to decrease the ionization suppression of peptides prior to MALDI MS analysis. ACKNOWLEDGMENT The authors thank Dr. Gary Case at the University of Wisconsin Biotechnology Center Peptide Synthesis Facility for helpful discussions on the net charge calculation of peptides. We wish to thank the UW School of Pharmacy Analytical Instrumentation Center for access to the MALDI FTMS instrument. This work was supported in part by the School of Pharmacy and Wisconsin Alumni Research Foundation at the University of Wisconsin-Madison, a National Science Foundation CAREER Award (Grant CHE-0449991), and National Institutes of Health through Grant 1R01DK071801. L.L. acknowledges an Alfred P. Sloan Research Fellowship. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review February 24, 2008. Accepted May 22, 2008. AC800382T (59) Castagnola, M.; Rossetti, D. V.; Corda, M.; Pellegrini, M.; Misiti, F.; Olianas, A.; Giardina, B.; Messana, I. Electrophoresis 1998, 19, 2273–2277.

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